The wall of blood vessels is made up of three concentric strata or tunicae (the tunica intima, media and adventitia) with well distinguished structure and composition. The tunica intima is made up of a single layer of endothelial cells supported on a basement membrane rich in collagen and proteoglycans and separated from the media by the internal elastic lamina. The tunica media is formed by vascular smooth muscle cells (VSMCs) and extracellular matrix, and the adventitia, the outermost layer, is essentially made up of connective tissue and fibroblasts.
The VSMCs are physiologically located in the tunica media. However, VSMCs can be found in the intimal layer as a result of a lack of organization of the structure of the vascular wall due to vascular pathologies or lesions such as atherosclerosis, hyperplasia of the tunica intima.
Intimal hyperplasia is a change in the vascular structure which occurs as a result of the biological repair mechanisms after a vascular lesion, either mechanical, surgical, inflammatory or immunological. The most characteristic finding of this structural change is the thickening of the intimal layer, due both to an increase in the number of cells and to an increase in the synthesis of extracellular matrix in which these cells are found (Davies M G, Hagen P O. Pathobiology of intimal hyperplasia. British Journal of Surgery 1994; 81:1254-1269). Ultimately, this process results in narrowing or stenosis of the vascular lumen.
There are various pathological situations associated with intimal hyperplasia and the triggering factors can generally be grouped together as physical lesions (in many cases iatrogenic lesions due to vascular surgery), inflammatory lesions (as in atherosclerotic lesions) or due to an increase of the tension of the wall (as in the case of pulmonary hypertension or the use of vein grafts in by-pass surgery). Some of the diseases associated with intimal hyperplasia include:
Late By-Pass Occlusion:
Revascularization with vein grafts is the standard treatment for occlusive arterial diseases when the occluded segment is large and an endarterectomy cannot be performed. The most used technique is the autogenic transplant of a segment of the saphenous vein, and it is used both in coronary surgery and in peripheral vascular surgery (intermittent claudication, thromboangiitis obliterans of the tibial artery, etc.). Although the procedure has a very good immediate result, in the long term, the vein graft suffers chronic maladaptive response to an arterial environment in which the primary component is the uncontrolled proliferation of vascular smooth muscle cells, giving rise to intimal hyperplasia which can later become complicated due to the development of atherosclerosis and thrombosis (Murphy G J, Angelini G D. Cardiovasc Ultrasound 2004; 21:2-8). The possibility of handling ex vivo vein transplants prior to their implantation using pharmacological methods, gene therapy or the application of synthetic coatings is an alternative that has been attempted to be used to prevent vascular graft failure (Mann M J, Whittermore A D, Donaldson M C, et al., Lancet 1999; 354: 1493-8; Bhardwaj S, Roy H, Ylä Herttuala, Expert Rev Cardiovasc Ther 2008; 6:641-52) and which in some cases has proven to be effective in experimental animals.
Post-Transplant Coronary Vasculopathy:
Post-transplant coronary vasculopathy is the main factor limiting long-term survival after a heart transplant. It manifests as an especially aggressive form of coronary artery disease that is different from conventional arteriosclerosis, which is caused by the combination of physical, chemical and immunological factors causing an endothelial lesion which in turn triggers the proliferation of vascular smooth muscle cells and intimal hyperplasia The pathological analysis of the lesions shows a thickening of the intima in which undifferentiated cells of the smooth muscle and macrophages and lymphocytes participate. This thickening of the intima leads to the obstruction of the coronary arteries, which ultimately leads to graft failure (Aranda and Hill, Chest 2000, 118(6): 1792-1800; Schmauss and Weis, Circulation (2008) 117(16):2131-41M: Weiss et al., Front Biosci. (2008) 13:2980-8).
An important aspect of the pathogenesis of vasculopathy is the interaction of immunological and non-immunological factors. In fact, for many years it was believed that intimal hyperplasia was due exclusively to immunological factors. However, immunosuppressant therapies have not been proven to be capable of reducing its incidence. On the contrary, an increase of hyperplasia after the introduction of treatments based on the use of immunosuppressants, such as cyclosporine, has been observed. In studies conducted with experimental animal models, promising results have been obtained when these immunosuppressant agents are combined with drugs such as MMF (mycophenolate mofetil) which inhibits DNA synthesis and therefore has a generalized antiproliferative effect that extends beyond the immune cells. Although revascularization procedures (percutaneous coronary angioplasty, coronary atherectomy, coronary bypass surgery or stent implantation) can be used in patients presenting localized stenoses, the diffuse character of arteriopathy makes the use of these therapies limited; therefore the development of new pharmacological therapies targeted at controlling the factors which determine intimal hyperplasia in this vasculopathy is necessary.
Post-Transplant Chronic Nephropathy:
The factors triggering coronary vasculopathy are also present in other post-transplant vasculopathies, as is the case of post-transplant nephropathy, which again represents the most frequent cause of renal transplant failure.
In addition to those mentioned, there are other diseases resulting from intimal hyperplasia. In order to identify when a disease is due to intimal hyperplasia, the person skilled in the art will use imaging techniques. Specifically, coronary angiography is used in the coronary tree, injecting contrasting fluid into the ostium of said arteries. Likewise, the 64-slice multislice CT and cardiac resonance angiography are also able to display atherosclerotic lesions in the coronary circulation.
Angiographic techniques are complemented with ultrasound techniques, such as intravascular ultrasound, which allow displaying the thickness of the arterial intimal layer, and determining if there is an atherosclerotic lesion and its characteristics. In normal conditions, the thickness of the intima cannot be measured microscopically because it is a single layer of cells. However, any measurable thickness (which is greater than a single cell layer) is pathological (FIG. 1).
There are other techniques, such as virtual histology, which allows determining the presence of lipids, calcium, a clot, fibrous tissue and hyperplasia, and OCT (optical coherence tomography), which uses laser technology to determine the entire thickness of the vascular wall and thus identifying the areas of intimal hyperplasia (see FIG. 2). Both in human arterial specimens and in experimental samples of animal models, morphometry is used for the histological analysis of the thickness of the various layers of the arterial wall. After the suitable treatment period, the animal will be euthanized and the studied vessels extracted and fixed for their subsequent analysis. Histological slices will be obtained and stained with hematoxylin-eosin. The internal elastic lamina will be identified and the areas of the various vascular layers evaluated (Gallo et al., 1998 Circulation 97:581-588)
Given the incidence and severity of these pathologies, there is a need to develop a tool which limits or prevents intimal hyperplasia and the pathological situations resulting from it, especially after surgical interventions or transplant surgery.
The inventors have surprisingly found that the 1.3 voltage-activated potassium channel (Kv1.3) blockers significantly limit intimal hyperplasia.
Traditionally, inhibitors of Kv1.3 have been used in therapies against immunological diseases, such as encephalomyelitis or multiple sclerosis (Wulff, H. et al., 2003. Curr. Opin. Drug Discov. Devel. 6: 640-647. Cahalan, M. D., et al., 2001. J. Clin. Immunol. 21:235-252). Kv1.3 was first identified in T-cells (Hu et al., The Journal of Immunology, 2007, 179: 4563-4570. Grissmer et al., Proc. Nat. Acad. Sci. 87: 9411-9415, 1990). In these cells, the Kv1.3 channel is tetra-homomeric, whereas in other types of cells, Kv1.3 is associated with subunits of other voltage-activated channels of the Kv1 family to form heteromeric potassium channels (Hu et al., The Journal of Immunology, 2007, 179: 4563-4570). As a consequence, the regulation of K+ flows through the membrane of the cells is often the combinatorial result of multiple molecular signaling pathways. The importance of Kv1.3 in immune system-related diseases has been proven in several studies.
Document WO2008088422 discloses the use of peptide inhibitors of Kv1.3 for the treatment of autoimmune diseases, allergies, diabetes and obesity and describes the manufacture and purification of derivatives of the toxins ShK, MgTx1, MTX1, HsTxl, wGVIA, HaTxl, etc. According to the data shown, the peptide derivatives of ShK are able to inhibit the human Kv1.3 current with an IC of −150 pM. ShK is, furthermore, a potent inhibitor of the proliferation of T-cells so their use in therapies for the treatment of immunological diseases such as multiple sclerosis, rheumatoid arthritis, dermatitis, diabetes type I, etc., is proposed.
K+ Channels in the Vascular Smooth Muscle
Potassium channels also play a very relevant role in the immediate and long-term regulation of the function of the vascular smooth muscle cells. The VSMCs of the walls of the vessels are cells which express a unique repertoire of contractile proteins, ion channels and signaling molecules aimed at maintaining vascular tone. At least four types of K+ channels have been identified in VSMCs: voltage-dependent K+ channels (Kv), such as Kv1.2, Kv1.3, Kv1.5, Kv1.6 and Kv2.1, calcium-activated K+ channels (such as the maxiK or BK channels), of inward-rectifying K+ channels (such as the KIR channels and KATP channels) and two-pore K+ channels, which are responsible for the background currents and the TASK and TWIK channels (reviewed in Jackson W F, Microcirculation 12, 113-127, 2005). Furthermore, the existence of regulating subunits of these channels together with the variation in their expression depending on the vascular bed, contributes to the fine regulation of the smooth muscle physiology. Thus, the pathological processes can be associated with the deregulation of multiple control mechanisms acting in parallel or in combination. Determining which mechanism leads to a specific vascular disease is an arduous scientific experiment task.
According to the foregoing, and due to the diffuse and distal location of hyperplastic lesions, the field of the art cannot provide effective therapies for the treatment of intimal hyperplasia. It would therefore be convenient to develop a therapeutic tool aimed at controlling the factors determining hyperplasia of the tunica intima and which is able to stop the migration and proliferation of the VSMCs therein, as well as the secretion of components of the cell matrix.